Hen Egg White Lysozyme (HEWL) Confers Resistance to Verticillium Wilt in Cotton by Inhibiting the Spread of Fungus and Generating ROS Burst

Verticillium wilt is a soil-borne vascular disease caused by the fungal pathogen Verticillium dahliae. It causes great harm to upland cotton (Gossypium hirsutum) yield and quality. A previous study has shown that Hen egg white lysozyme (HEWL) exerts strong inhibitory activity against V. dahliae in vitro. In the current study, we introduced the HEWL gene into cotton through the Agrobacterium-mediated transformation, and the exogenous HEWL protein was successfully expressed in cotton. Our study revealed that HEWL was able to significantly inhibit the proliferation of V. dahlia in cotton. Consequently, the overexpression of HEWL effectively improved the resistance to Verticillium wilt in transgenic cotton. In addition, ROS accumulation and NO content increased rapidly after the V. dahliae inoculation of plant leaves overexpressing HEWL. In addition, the expression of the PR genes was significantly up-regulated. Taken together, our results suggest that HEWL significantly improves resistance to Verticillium wilt by inhibiting the growth of pathogenic fungus, triggering ROS burst, and activating PR genes expression in cotton.


Introduction
Cotton disease is one of the most important factors causing the decline in cotton yield and quality.In particular, Verticillium wilt results in a severe yield reduction in cotton.In China, the primary pathogen causing the Verticillium wilt of cotton is V. dahliae, which can also infect about 660 plant species belonging to 38 families, including Solanaceae, Fabaceae, Rosaceae, Brassicaceae and Asteraceae [1,2].In the beginning, V. dahliae derived from the soil starts colonizing cotton roots and enters the vascular structure.The vascular system produces a large number of mycelia and conidia which proliferate and spread to other plant parts.Ultimately, it damages the plants by blocking the vascular system and releasing toxic proteins [2,3].However, the complete infestation progress is not completely understood to date.The vascular tissues of plants can transmit pathogens, although this is not the only way [2].Verticillium wilt is a typical vascular disease that can affect crops for a long time and is extremely difficult to cure once infected [4].Currently, no highly disease-resistant cotton varieties are available in China as resources.Therefore, improving cotton varieties to increase disease resistance and reduce the impact of Verticillium wilt on cotton yield is a significant challenge in the cotton breeding industry.
Biological breeding is an essential technology for the rapid and directional breeding of new crop varieties.Bacillus thuringiensis (Bt) cotton has been successfully commercialized widely for many years, and it effectively resolves the threat of bollworm infestation to cotton yield [5].In recent years, several studies have shown that the overexpression of foreign genes in cotton can improve the resistance to Verticillium wilt to some extent.The ectopic expression of plant defensin NaD1 from Nicotiana alata, harpin protein Hpa1Xoo from Xanthomonas oryzae, and cell surface-localized immune receptor Ve1 from Solanum lycopersicum show enhanced tolerance to Verticillium wilt in cotton [6][7][8].A fusion protein of the antifungal peptide BbAFP1 and the ergosterol domain of ergosterol targeting the fungal cell wall and cell membrane are expressed in cotton, which show higher disease resistance compared to the expression of BbAFP1 alone [9].In addition, the overexpression of endogenous genes derived from cotton can also improve its resistance to Verticillium wilt.Overexpression of GhMYB4 (MYB transcription factor), GhPFN2 (profilin gene), GhDIR1 (dirigent1 gene), and GbRLK (receptor-like kinase gene) in cotton improve the defense response of plants to pathogens [10][11][12].
The plant defense responses mainly include constitutive defense system, inducible defense (PAMP-triggered immunity, PTI, and effector-triggered immunity, ETI), and systemicacquired resistance (SAR) [13][14][15][16].Salicylic acid (SA) and jasmonic acid (JA) are important signal molecules in the SAR signal pathway.Gbvdr6 from the Verticillium wilt-resistant cotton Hai7124 and NPR1 from Arabidopsis confer resistance to Verticillium wilt in cotton through the regulation of the JA/ET and SA signaling pathways [17,18].Previously, many cotton resistance-related genes have been identified.For instance, plant cell wall-associated receptor-like kinases GhWAKL, pectin methylesterase-inhibiting protein GhPMEI3, ribosomal protein GhRPS6, and laccase gene GhLAC15 are shown to play essential roles in cotton resistance to pathogen infection [19][20][21][22].During V. dahliae infection in cotton plants, several transcription factors are also involved in regulating plant disease resistance.BEL1-Like TF GhBLH7-D06 and HD-ZIP I TF GhHB12 negatively regulate cotton resistance to Verticillium wilt [23,24], while R2R3-type MYB TF GhODO1 and WRKY TF GhWRKY1-like are known to positively regulate cotton resistance to Verticillium wilt [25,26].
Lysozyme is highly abundant in egg white, accounting for about 3-4% of the total protein content [27].Hen egg white lysozyme (HEWL) is a c-type lysozyme that hydrolyzes β-1,4 glycosidic bonds between the N-acetylmuramic acid (NAM) and N-acetyl glucosamine (NAG) of peptidoglycan in the bacterial cell wall [28].Further, some studies have also shown the antifungal activity of HEWL.Our previous study indicated that HEWL could effectively inhibit the growth of fungal pathogens, V. dahliae and F. oxysporum, and that the antifungal rate was as high as 75.7% compared with phosphate buffer (pH6.2) [29].In this study, the HEWL gene driven by the plastocyanin promoter (PetE Pro) was transformed into G. hirsutum R15 using the Agrobacterium-mediated method.We observed that HEWL confers resistance to Verticillium wilt in cotton under greenhouse and field conditions.HEWL transgenic cotton exhibited ROS burst and increased the expression of pathogenesis-related (PR) genes expression under biotic stress.The V. dahliae biomass was also lower in transgenic cotton than in wild-type cotton.Altogether, our results suggested that that constitutive expression of HEWL in cotton could increase resistance to Verticillium wilt.

Generation of HEWL Transgenic Cotton Plants
The plant expression vector pBI121-35S::CP4_EPSPS-PetE::HEWL was shown in Figure 1a.PetE promoter from plastocyanin gene 5 UTR of Medicago sativa was used upstream of HEWL ORF.Moreover, the expression of glyphosate-resistant gene CP4-EPSPS as a selectable marker gene in the vector was essential in selecting transgenic plants.The plant expression vector was introduced into upland cotton R15 via the Agrobacteriummediated transformation method.The process of cotton genetic transformation went through co-cultivation formation, the generation of primary callus, embryogenic callus, embryoid body formation, and finally differentiation into regenerated plants, which were grafted into 25-day Hai 7124 (Figure 1b).The exogenous genes CP4-EPSPS and HEWL were identified by PCR in the regenerated cotton plants (Figure S2).Three homozygous lines T111-16, T58-22, and T1008-6 were identified through three generations of continuous selfing, and the target genes were detected in all plants of homozygous transgenic lines which showed resistance to glyphosate (Figure S3).Southern blot analysis revealed a single copy insertion of the exogenous gene in lines T111-16 and T58-22 (Figure 1c).qPCR confirmed the high expression of HEWL in the roots, stems, and leaves of transgenic cotton, and the expression level of line T58-22 was higher than that of line T111-16 and T1008-6 (Figure 1d).Further, Western blot analysis confirmed the expression of HEWL protein in the transgenic cotton.The results showed that the expression level of HEWL in T58-22 was higher than that in line T111-16 (Figure 1e), which was consistent with the qPCR results.
identified by PCR in the regenerated cotton plants (Figure S2).Three homozygous lines T111-16, T58-22, and T1008-6 were identified through three generations of continuous selfing, and the target genes were detected in all plants of homozygous transgenic lines which showed resistance to glyphosate (Figure S3).Southern blot analysis revealed a single copy insertion of the exogenous gene in lines T111-16 and T58-22 (Figure 1c).qPCR confirmed the high expression of HEWL in the roots, stems, and leaves of transgenic cotton, and the expression level of line T58-22 was higher than that of line T111-16 and T1008-6 (Figure 1d).Further, Western blot analysis confirmed the expression of HEWL protein in the transgenic cotton.The results showed that the expression level of HEWL in T58-22 was higher than that in line T111-16 (Figure 1e), which was consistent with the qPCR results.

HEWL Expression Improved Resistance to Verticillium Wilt in Transgenic Cotton
We assessed the resistance of HEWL transgenic cotton to Verticillium wilt in the greenhouse.After inoculation with Vd991, transgenic cotton lines T58-22 and T111-16 that were growing normally and only had a small number of yellow spots, showed strong resistance to Verticillium wilt, while the leaves of wild-type cotton began to wither and fall off (Figure 2a).The disease indexes (DIs) of HEWL transgenic cotton lines were significantly lower than that of WT at 10 days, 20 days, and 30 days after inoculation with Vd991.The DI of the three lines was less than 30 and showed significant the resistance to the Vd991 strain (Figure 2b).The fungal biomass determination revealed that HEWL transgenic cotton lines was lower than fungal biomass in WT after inoculation (Figure 2c).Further, the trypan blue staining showed that V. dahliae caused the death of leaf cells in wild-type plants (more blue staining area), while the leaves of HEWL transgenic plants only had a smaller number of cells (Figure 2d).

HEWL Expression Improved Resistance to Verticillium Wilt in Transgenic Cotton
We assessed the resistance of HEWL transgenic cotton to Verticillium wilt in the greenhouse.After inoculation with Vd991, transgenic cotton lines T58-22 and T111-16 that were growing normally and only had a small number of yellow spots, showed strong resistance to Verticillium wilt, while the leaves of wild-type cotton began to wither and fall off (Figure 2a).The disease indexes (DIs) of HEWL transgenic cotton lines were significantly lower than that of WT at 10 days, 20 days, and 30 days after inoculation with Vd991.The DI of the three lines was less than 30 and showed significant the resistance to the Vd991 strain (Figure 2b).The fungal biomass determination revealed that HEWL transgenic cotton lines was lower than fungal biomass in WT after inoculation (Figure 2c).Further, the trypan blue staining showed that V. dahliae caused the death of leaf cells in wildtype plants (more blue staining area), while the leaves of HEWL transgenic plants only had a smaller number of cells (Figure 2d).Further, we conducted field disease resistance experiments in Anyang, Henan Province, and Langfang, Hebei Province.In Anyang, we observed that most leaves of WT plants had light yellow patches on between leaf margins and leaf veins, which gradually expanded, and the leaves started to fall off the bottom of the plant (Figure 3a).The DI statistics in September revealed that the DI of HEWL transgenic cotton was significantly different from that of WT and the susceptible control "Jimian11".In particular, the DIs of lines T58-22 and T111-16 were lower than 15, showing high disease resistance (HR) (Figure 3b).Also, HEWL transgenic cotton showed a better disease resistance phenotype (Figure 3c), and their DIs were all lower than 15, exhibiting high resistance in Langfang (Figure 3d).Altogether, these results indicated that the disease resistance was significantly improved in HEWL transgenic cotton.
Further, we conducted field disease resistance experiments in Anyang, Henan Province, and Langfang, Hebei Province.In Anyang, we observed that most leaves of WT plants had light yellow patches on between leaf margins and leaf veins, which gradually expanded, and the leaves started to fall off the bottom of the plant (Figure 3a).The DI statistics in September revealed that the DI of HEWL transgenic cotton was significantly different from that of WT and the susceptible control "Jimian11".In particular, the DIs of lines T58-22 and T111-16 were lower than 15, showing high disease resistance (HR) (Figure 3b).Also, HEWL transgenic cotton showed a better disease resistance phenotype (Figure 3c), and their DIs were all lower than 15, exhibiting high resistance in Langfang (Figure 3d).Altogether, these results indicated that the disease resistance was significantly improved in HEWL transgenic cotton.

HEWL Transgenic Cotton Enhances Disease Resistance by Generating ROS Burst and Activating PR Genes
In the current study, ROS burst was observed in HEWL transgenic cotton leaves infected with V. dahliae.We observed that the leaves of lines T58-22 and T111-16 rapidly showed reddish-brown precipitation after inoculation with V. dahliae, which was different from the phenotype of WT leaves, as detected by 3,3 -diaminobenzidine tetrahydrochloride (DAB) staining (Figure 4a).Further, we examined the H 2 O 2 content in leaves after inoculation with V. dahliae and found that the H 2 O 2 content of HEWL transgenic cotton was significantly higher than that of WT.In WT, the H 2 O 2 content gradually increased and reached a peak at 5 h, while in HEWL transgenic cotton, it increased sharply with two peaks at 1 h and 5 h (Figure 4b).The NO content in HEWL transgenic cotton increased immediately and began to gradually decrease after inoculation with V. dahliae (Figure 4c).These results suggested that an immediate ROS burst was triggered in the transgenic cotton lines in response to biotic stress, activating hormonal signaling pathways.

HEWL Inhibits the Growth of V. dahliae in Cotton
Our previous study showed that HEWL could effectively inhibit the growth of V. dahliae with a bacteriostatic rate of 75.7% [29].The in vitro fungal inhibition test showed that HEWL could effectively inhibit the growth of V. dahliae.Also, the protein from HEWL transgenic cotton could inhibit the proliferation of V. dahliae.The proliferation of V. dalliae in non-transgenic WT cotton was the same as that of the negative control (Figure 5a).We inoculated cotton plants with Vd991-GFP to observe the strength of green fluorescence in plants.After 20 days of inoculation, a strong GFP signal was observed in the stem of WT cotton, whereas a weaker GFP fluorescence was observed in the transgenic cotton plants HEWL was able to activate expression of PR genes in cotton.The charpin-induced 1 (HIN1) and HSR203J were the HR marker genes of hypersensitivity [30,31].Previous studies demonstrated that the cotton profilin gene GhPFN2 was involved in plant innate immune responses [32].When plants were attacked by fungal pathogens, the PDF1.2 gene encoding plant defensins was regulated by simultaneous jasmonate and ethylene signaling [33].The NPR1 (non-expressor of PR gene) gene was a key regulator of SA-mediated SAR and played an important role in plant basic defense [34].Nitric oxide-associated protein 1 (NOA1) was involved in various abiotic stress responses and required for plants to resist pathogen infection [35].After inoculation with V. dahliae, qPCR analysis revealed that the expression of HIN1 and HSR203J was significantly up-regulated in the HEWL transgenic lines than the WT (Figure 4d,e).In addition, the PR genes GhPFN2, GhPDF1.2,GhNPR1, and GhNOA1 were also significantly up-regulated in HEWL transgenic lines (Figure 4f-j).These results suggested that HEWL transgenic cotton responded to biotic stress by regulating the expression of PR genes.

HEWL Inhibits the Growth of V. dahliae in Cotton
Our previous study showed that HEWL could effectively inhibit the growth of V. dahliae with a bacteriostatic rate of 75.7% [29].The in vitro fungal inhibition test showed that HEWL could effectively inhibit the growth of V. dahliae.Also, the protein from HEWL transgenic cotton could inhibit the proliferation of V. dahliae.The proliferation of V. dalliae in non-transgenic WT cotton was the same as that of the negative control (Figure 5a).We inoculated cotton plants with Vd991-GFP to observe the strength of green fluorescence in plants.After 20 days of inoculation, a strong GFP signal was observed in the stem of WT cotton, whereas a weaker GFP fluorescence was observed in the transgenic cotton plants (Figure 5b).These results indicated that the overexpression of HEWL can effectively inhibit the growth of V. dahliae in cotton.(Figure 5b).These results indicated that the overexpression of HEWL can effectively inhibit the growth of V. dahliae in cotton.

Fungal Inhibitory Activity of HEWL
The overexpression of PR genes and antimicrobial peptide (AMP) genes in plants can improve plant resistance to pathogens.In addition, the precise editing and knockout of (a) Antifungal activities of HEWL transgenic cotton against V. dahliae Vd991 on PDA plates.1) 0.5 mg/mL HEWL from egg white; 2) 1 mg/mL HEWL from egg white; 3) ddH 2 O; 4) 1 mg/mL total protein from HEWL transgenic cotton line T111-16; 5) 1 mg/mL total protein from HEWL transgenic cotton line T58-22; 6) 1 mg/mL total protein from non-transgenic cotton plant R15.(b) In situ observation of V. dahliae with GFP of HEWL transgenic cotton after V. dahliae inoculation.Wild type: non-transgenic cotton plant R15; NT-wild type: non-treatment wide type cotton plant.Scale bars = 100 µm.All experiments were repeated at least three times.

Fungal Inhibitory Activity of HEWL
The overexpression of PR genes and antimicrobial peptide (AMP) genes in plants can improve plant resistance to pathogens.In addition, the precise editing and knockout of target genes using CRISPR/Cas9 systems can also enhance the disease tolerance of the plant.However, very few antifungal proteins are known to impart disease resistance in crop plants.HEWL can hydrolyze the cell wall of Gram-positive bacteria to generate a bacteriostatic effect [28].Our previous study demonstrated that HEWL can effectively inhibit growth of V. dahliae and F. oxysporum [29].This study also confirmed that the HEWL inhibited the growth of V. dahliae (Figure 5a).However, the mechanism by which it inhibits fungal growth remains unclear.Düring et al. found that the α-helix at the C-terminal of T4 lysozyme has "non-enzymatic antibacterial activity", which interferes with the cell membranes of bacteria and fungi [36].HEWL may also have the same non-enzymatic activity.Destabilase-Lysozyme (mlDL) from Hirudo medicalis has a nonenzymatic antibacterial effect independent of its muramidase activity [37,38].Therefore, the mechanism of HEWL inhibiting fungi, including enzymatic and non-enzymatic activity, needs to be further studied and determined.

HEWL Produces a ROS Burst in Transgenic Cotton
Pathogenic fungi produce oxidative bursts shortly after infecting plant tissues, characterized by the transient overproduction of ROS [39].ROS usually refers to H 2 O 2 , O 2 -, OH, O 2 , and NO also belongs to ROS in a sense [40].Oxathiapiprolin is a plant-specific pathogenic oomycete inhibitor.In a previous study, pathogen-infected Arabidopsis thaliana plants were treated with oxathiapiprolin to enhance H 2 O 2 accumulation [41].Cassava (Manihot esculenta Crantz) AP2/ERF transcription factor MeRAVs positively co-regulate cassava bacterial blight (CBB) resistance and stimulate innate immune responses by modulating reactive oxygen species (ROS) burst [42].Hcm1 transgenic cotton also generated a ROS burst after V. dahliae inoculation [43].This study exhibited that after V. dahliae inoculation, the H 2 O 2 and NO contents reached the peak within 1 h in HEWL transgenic cotton, while it reached the peak at 5 h in the WT plants (Figure 4a-c).In this study, we found that after V. dahliae infection, a ROS burst was triggered in cotton, which accumulated more rapidly in transgenic plants than in the wild-type plants.

HEWL Elicits Defense Responses against Pathogens in Cotton Plants
In the greenhouse, HEWL transgenic cotton plants showed stronger disease resistance and significantly decreased fungal biomass after V. dahlia inoculation (Figure 2a-c).In vitro experiments revealed that HEWL was able to effectively inhibit the growth of V. dahliae, and the protein extracted from HEWL transgenic cotton could also inhibit fungal growth (Figure 5a).Compared to the wild type, HEWL transgenic cotton plants showed strong resistance to Verticillium wilt and significantly improved disease resistance index in the field (Figure 3).These results indicated that overexpression of HEWL in cotton could effectively improve the Verticillium wilt resistance by inhibiting fungal growth.In addition, overexpression of HEWL in cotton can activate the expression of the disease-resistance PR genes (Figure 4d-j).Our results indicated that HEWL transgenic cotton responded to biotic stress by regulating the expression of disease resistance-related genes.Plants evoke innate immunity against pathogen challenge upon recognizing chitin in the fungal cell wall.Fujikawa et al. showed that α-1,3-glucan protected the fungal cell wall to delay innate immune defense responses [44].Taken together, overexpression of HEWL in cotton can elicit defense responses against pathogens by hydrolyzing fungal cell walls.

The Strategies and Future Prospects of Controlling Cotton Diseases
There are many approaches to controlling cotton diseases, such as seed disinfection, crop management, biological control, and the breeding of resistant varieties.Environmentally friendly and ecologically safe biocontrol methods are an important strategic requirement for sustainable agricultural development.Many microorganisms have been proven to have biocontrol effects, such as Pseudomonas spp.and Serratia plymutica [45].In addition, endophytic fungi isolated from cotton roots can also improve the resistance to Verticillium wilt [46].However, there is no effective biological agent to control cotton disease in the market.Long-term production practice shows that biological breeding is the fundamental measure of disease control in the world.The overexpression of antimicrobial proteins and PR genes can effectively improve plant disease resistance, but the available variety for production by transgenic method must undergo the safety assessment of GM crops according to Chinese legal regulations, which is time-consuming, expensive, and complex.Recently, researchers have shown created transgene-free edited cotton lines through editing the Gh14-3-3d gene using CRISPR/Cas9 method to improve resistance against V. dahliae invasion [47].Next, we will use CRISPR/Cas9 technology to develop transgene-free edited plants from transgenic lines, which is a key issue in the improvement of the breeding of transgenic disease-resistant crops.

Plant Materials and Growth Conditions
The cotton wild type varieties Gossypium hirsutum L. R15 and Gossypium barbadense L. Hai 7124 were provided by the Cotton Research Institute, Shanxi Academy of Agricultural Sciences.The plants were grown in a growth chamber at 25 • C under 16 h light/8 h dark conditions in Chinese Academy of Agricultural Sciences.Moreover, the medium for cotton tissue culture was previously described by Wu et al. [48], and the cotton genetic transformation materials were cultured in a light incubator (16 h light/8 h dark) at 28 • C.

V. dahliae Strain and Culture Conditions
V. dahliae strains Vd991 and Vd991-GFP (harboring GFP gene) were provided by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.The fungal strains were cultured on potato dextrose agar (PDA) medium at 25 • C for 7 days.Next, the colonies were inoculated into Czapek medium [49], and shocked at 200 rpm for 7 days at 25 • C. The conidia were counted using a hemocytometer, and their final concentration was adjusted to 5 × 10 6 mL −1 with deionized water for infection, as described previously [50].

Cotton Transformation and Transgenic Plant Selection
The antifungal gene HEWL and glyphosate resistance gene CP4-EPSPS were synthesized by GenScript (Nanjing, China).We added a signal peptide from alpha-amylase (26 amino acids) to the N terminal of HEWL.The sequences of the genes were shown in Figure S1.We constructed the plant expression vector pBI121-35S::CP4_EPSPS-PetE::HEWL, which harbored the HEWL and CP4-EPSPS genes.The sequences of corresponding primer pairs were found in Table S1.We introduced the vector into Agrobacterium tumefaciens strain LBA4404 via electroporation.The Agrobacterium carrying vector pBI121-35S::CP4_EPSPS-PetE::HEWL was inoculated in LB liquid medium (50 mg/L Kan, 50 mg/L Rif) and cultured at 200 rpm for 48 h at 28 • C. The cell suspension was diluted with MS liquid medium to OD600 0.4 for a subsequent experiment.Hypocotyls of upland cotton R15 sterile seedlings were infected using the Agrobacterium-mediated method [48] and were cultured under light conditions (16 h light/8 h dark) at 28 • C in an incubator.The transgenic seedlings obtained by tissue culture were grafted to the rootstock Gossypium barbadense L. Hai 7124 and cultivated in the greenhouse.

PCR and qPCR
The genomic DNA of cotton leaf was extracted using the CTAB method [51].Using the isolated DNA as a template, the target genes, CP4-EPSPS and HEWL, were amplified.PCR amplification was performed according to Guo et al. [52].
The total RNA of cotton tissue was extracted using the plant RNA extraction kit (Sangon Biotech, Shanghai, China).cDNA was synthesized using the reverse transcription kit EasyScript ® All-in-One First-Strand cDNA Synthesis SuperMix (TransGen, Beijing, China).Using cDNA as a template, qPCR was performed using TransStart ® Tip Green qPCR SuperMix Kit (TransGen, Beijing, China) on the ABI Prism 7500 real-time PCR platform (Applied Biosystems, Foster City, CA, USA).qPCR amplification program was as follows: 95 • C for 10 min; 95 • C for 10 s; and 60 • C for 30 s for 40 cycles of amplification.Three biological replicates and three technical replicates were performed for each sample.Each biological replicate was obtained from three plants of each line as one sample.PCR and qPCR-related gene primers were shown in Table S1.

Southern Blot and Western Blot
For Southern blot analysis, 30 µg gDNA from the young leaves of HEWL transgenic cotton and WT was extracted and digested with EcoRI or NdeI, respectively.We labelled the CP4-EPSPS gene as a probe via PCR with DIG Probe Synthesis Kit (Roche Applied Science, Mannheim, Germany), and the primer pairs (CP4-F3, CP4-R3) were shown in Table S1.The DNA electrophoresed on the gel was transferred to a nylon membrane.Southern blotting was performed using DIG High Prime DNA Labeling and Detection Starter Kit II (Roche Applied Science, Mannheim, Germany).The DIG-labeled DNA Molecular-Weight Marker was also obtained from Roche Applied Science.
The Western blot analysis was conducted as described by Jorgensen et al. [53].Total plant protein was extracted from HEWL transgenic and WT cotton leaves [54].After electrophoresis on 15% SDS-PAGE, the protein was transferred to a nitrocellulose membrane (20 V, 10 h) and blocked with a blocking solution at 25 • C for 2~3 h.Next, the antirabbit HEWL antibody (ABclonal, Wuhan, China) and goat anti-rabbit IgG-AP (Abcam, Cambridge, UK) was added.The dye was BCIP/NBT Alkaline Phosphatase reagent (Solarbio, Beijing, China).

Evaluation of Cotton Resistance to Verticillium Wilt
Cotton seeds were sown in nursery trays and grown normally for 1 month under greenhouse conditions.One-month-old cotton seedlings were inoculated using V. dahliae spore suspension using the root irrigation method [55].Each cotton plant was treated with 20 mL conidial suspension (5 × 10 6 conidia/mL).We treated at least 30 plants per line.The infection assay was repeated at least four times in the greenhouse.We observed the incidence of cotton and counted the disease index.Vd991-GFP was used to infect onemonth-old cotton seedlings using the root irrigation method under greenhouse conditions.After 1 month, the plants showed disease symptoms.Then, the plant roots were collected and sliced for sample preparation for subsequent microscopic observation.Samples were observed using a confocal microscope (Zeiss LSM700, Jena, Germany).Each sample was taken from three treated plants of each line.
The field disease resistance was evaluated in V. dahliae disease-field of the Institute of Plant Protection of CAAS, Langfang, Hebei Province and the Institute of Cotton Research of CAAS, Anyang, Henan Province.The Jimian11 and 86-1varieties susceptible to Verticillium wilt were used as the susceptible control, and Zhongzhimian2 and ZhongzhimianKV3 were planted as the disease-resistant control.The experiment adopted 2 rows of planting with about 50 plants in each plot.Transgenic lines, non-transgenic WT, and the control were sown in rows with a complete block design with four replicates.The disease incidence was investigated in mid-September, and the disease index was counted.

Figure 2 .
Figure 2. Resistance to V. dahliae of HEWL transgenic cotton homozygous lines in the greenhouse.(a) Resistance phenotypes of transgenic cotton lines T58-22 and T111-16 in the greenhouse.(b) DIs of transgenic cotton homozygous lines.(c) Relative fungal biomass of transgenic cotton homozygous lines.(d) Trypan blue staining of leaves from transgenic cotton homozygous lines plants after V. dahliae inoculation.Scale bars = 5 mm.T58-22, T111-16, and T1008-6: transgenic cotton homozygous lines; WT (wild type): non-transgenic cotton plant R15; NT-wild type: non-treatment wild type cotton plant.Data were obtained from three independent experiments and represent the means ± SE; ** p < 0.01.

Figure 2 .
Figure 2. Resistance to V. dahliae of HEWL transgenic cotton homozygous lines in the greenhouse.(a) Resistance phenotypes of transgenic cotton lines T58-22 and T111-16 in the greenhouse.(b) DIs of transgenic cotton homozygous lines.(c) Relative fungal biomass of transgenic cotton homozygous lines.(d) Trypan blue staining of leaves from transgenic cotton homozygous lines plants after V. dahliae inoculation.Scale bars = 5 mm.T58-22, T111-16, and T1008-6: transgenic cotton homozygous lines; WT (wild type): non-transgenic cotton plant R15; NT-wild type: non-treatment wild type cotton plant.Data were obtained from three independent experiments and represent the means ± SE; ** p < 0.01.

Figure 3 .
Figure 3. HEWL improved resistance to Verticillium wilt of transgenic cotton in V. dahliae diseasefield.(a) Resistance phenotype of HEWL transgenic cotton in Anyang of Henan Province, China, in 2021.(b) The DIs of HEWL transgenic cotton in Anyang of Henan Province, China, in 2021.G. hirsutum cv.Zhongzhimain 2 as the resistance variety control; G. hirsutum cv.Jimian11 as susceptible variety control.(c) Resistance phenotype of HEWL transgenic cotton in Langfang of Hebei Province, China, in 2021.(d) The DIs of HEWL transgenic cotton in Langfang of Hebei Province, China, in 2021.G. hirsutum cv.Zhongzhimain KV3 as the resistance variety control; G. hirsutum cv.86-1 as susceptible variety control.T58-22, T111-16, and T1008-6: transgenic cotton homozygous lines; WT: non-transgenic cotton plant R15.Data were obtained from four independent experiments and represent the means ± SE.The letters (a,c) indicate significant differences at p ≤ 0.01 according to a randomization one-way ANOVA test.

Figure 3 .
Figure 3. HEWL improved resistance to Verticillium wilt of transgenic cotton in V. dahliae diseasefield.(a) Resistance phenotype of HEWL transgenic cotton in Anyang of Henan Province, China, in 2021.(b) The DIs of HEWL transgenic cotton in Anyang of Henan Province, China, in 2021.G. hirsutum cv.Zhongzhimain 2 as the resistance variety control; G. hirsutum cv.Jimian11 as susceptible variety control.(c) Resistance phenotype of HEWL transgenic cotton in Langfang of Hebei Province, China, in 2021.(d) The DIs of HEWL transgenic cotton in Langfang of Hebei Province, China, in 2021.G. hirsutum cv.Zhongzhimain KV3 as the resistance variety control; G. hirsutum cv.86-1 as susceptible variety control.T58-22, T111-16, and T1008-6: transgenic cotton homozygous lines; WT: non-transgenic cotton plant R15.Data were obtained from four independent experiments and represent the means ± SE.The letters (a,c) indicate significant differences at p ≤ 0.01 according to a randomization one-way ANOVA test.

Figure 4 .
Figure 4. ROS burst in leaves of HEWL transgenic cotton dipped in a conidial suspension of V. dahliae.(a) In situ observation of ROS in HEWL transgenic cotton leaves with DAB staining dipped in a conidial suspension of V. dahliae.At least four independent leaves were used for this experiment.Scale bars = 5 mm.(b) H2O2 content of HEWL transgenic cotton leaves dipped in a conidial suspension of V. dahliae.(c) NO content of HEWL transgenic cotton leaves dipped in a conidial suspension of V. dahliae.(d-j) Relative expression of the PR genes of HEWL transgenic cotton dipped in a conidial suspension of V. dahliae.Data were obtained from four independent experiments and represent the means ± SE; ** p < 0.01.

Figure 4 .
Figure 4. ROS burst in leaves of HEWL transgenic cotton dipped in a conidial suspension of V. dahliae.(a) In situ observation of ROS in HEWL transgenic cotton leaves with DAB staining dipped in a conidial suspension of V. dahliae.At least four independent leaves were used for this experiment.Scale bars = 5 mm.(b) H 2 O 2 content of HEWL transgenic cotton leaves dipped in a conidial suspension of V. dahliae.(c) NO content of HEWL transgenic cotton leaves dipped in a conidial suspension of V. dahliae.(d-j) Relative expression of the PR genes of HEWL transgenic cotton dipped in a conidial suspension of V. dahliae.Data were obtained from four independent experiments and represent the means ± SE; ** p < 0.01.